A New Generation of Gel-Forming Polysaccharides - ACS Symposium

Chapter 18

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A New Generation of Gel-Forming Polysaccharides An X-ray Study R. Chandrasekaran and V. G. Thailambal Whistler Center for Carbohydrate Research, Purdue University, West Lafayette, IN 47907 Using computer modeling, jointly with x-ray fiber diffraction data, the molecular architectures of two different gel-forming polysaccharides have been examined. Preliminary results indicate that the neutral and doubly branched capsular polysaccharide from Rhizobium trifolii can form a 2-fold single helix of pitch 1.96 nm or a half-staggered, 4-fold double -helix of pitch 3.92 nm. The molecules are likely to be stabilized by main chain -- side chain interactions. Detailed structure analysis reveals that the monovalent salt forms of gellan, an anionic, linear extracellular polysaccharide, exist as half-staggered, parallel double-helices containing 3-fold, left-handed polysaccharide chains of pitch 5.63 nm. The double -helix is stabilized by interchain hydrogen bonds involving the carboxylate groups. The crystal structure of the potassium salt shows that double-helix -- potassium -- water -- potassium -- double-helix interactions promote the aggregation of molecules and subsequent gelation. Extrapolation of these results by computer model building swiftly reveals the ability of calcium ions to establish direct and strong doublehelix -- calcium -- double-helix interactions. This explains the good gelling behavior of gellan at a very low calcium concentration. Further, modeling calculations enable us to understand that the poor gelling properties of native gellan are due to the presence of the glycerate, rather than acetate, groups. Computer modeling is an exciting and powerful technology for obtaining the three-dimensional structures of molecules. The process begins with a knowledge of the chemical structure. Based the fundamental principles of stereochemistry, the shapes of molecules, ranging from simple oligosaccharides to complex 0097-6156/90ΛΜ30-Ο300$06.00Α) © 1990 American Chemical Society

French and Brady; Computer Modeling of Carbohydrate Molecules ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

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c a r b o h y d r a t e s , can be i n v e s t i g a t e d . Computer modeling can f u r t h e r be used to examine, f o r example, p r o t e i n - p o l y s a c c h a r i d e , and n u c l e i c a c i d - d r u g , i n t e r a c t i o n s . A d e t a i l e d study o f the m o l e c u l a r a r c h i t e c t u r e s i s n e c e s s a r y i n o r d e r to understand the s t r u c t u r e f u n c t i o n - p r o p e r t y r e l a t i o n s h i p s . T h i s , i n t u r n , i s c r u c i a l t o the development o f many i n d u s t r i a l polymers and new b i o t e c h n o l o g i c a l p r o d u c t s . Aqueous s o l u t i o n s o f the r e c e n t l y d i s c o v e r e d b a c t e r i a l p o l y s a c c h a r i d e s such a s g e l l a n and r e l a t e d polymers, and the c a p s u l a r p o l y s a c c h a r i d e from Rhizobium trifolii (RTPS), e i t h e r d i s p l a y e x t r e m e l y h i g h v i s c o s i t y over a wide range o f temperature o r form g e l s o f e x c e l l e n t s t r e n g t h and t e x t u r e a t low concentrations. They are p a r t i c u l a r l y u s e f u l to the food i n d u s t r y . Interactions i n v o l v i n g the polymer c h a i n s , water m o l e c u l e s and c a t i o n s i n the case o f a n i o n i c polymers, t o g e t h e r w i t h those between the c r y s t a l l i n e ( o r d e r e d ) and n o n - c r y s t a l l i n e ( d i s o r d e r e d ) r e g i o n s o f the polymer specimens are r e s p o n s i b l e f o r the r h e o l o g i c a l b e h a v i o r o f the polymer s o l u t i o n s Q ) . X-RAY FIBER DIFFRACTION S t r u c t u r a l i n f o r m a t i o n a t the m o l e c u l a r l e v e l can be e x t r a c t e d u s i n g a number o f e x p e r i m e n t a l t e c h n i q u e s which i n c l u d e , but a r e not r e s t r i c t e d t o , o p t i c a l r o t a t i o n , i n f r a - r e d and u l t r a - v i o l e t s p e c t r o s c o p y , n u c l e a r magnetic resonance i n the s o l i d s t a t e and i n s o l u t i o n , d i f f r a c t i o n u s i n g e l e c t r o n s , n e u t r o n s o r x - r a y s . Not a l l o f them, however, are capable o f y i e l d i n g s t r u c t u r a l d e t a i l s t o the same d e s i r a b l e e x t e n t . By f a r , e x p e r i e n c e shows t h a t x-ray f i b e r d i f f r a c t i o n ( 2 ) , i n c o n j u n c t i o n w i t h computer model b u i l d i n g , i s the most p o w e r f u l t o o l which e n a b l e s t o e s t a b l i s h the s p a t i a l arrangement o f atoms i n polymer m o l e c u l e s . A l l t h r e e major c l a s s e s o f b i o p o l y m e r s — polysaccharides, p o l y p e p t i d e s and p o l y n u c l e o t i d e s , a s w e l l a s most s y n t h e t i c polymers, tend to adopt long h e l i c a l s t r u c t u r e s . Under t h e s e c i r c u m s t a n c e s , i t i s i m p o s s i b l e to produce s i n g l e c r y s t a l s s u i t a b l e f o r t r a d i t i o n a l x-ray c r y s t a l l o g r a p h i c i n v e s t i g a t i o n s , but o f t e n p o s s i b l e t o prepare p o l y c r y s t a l l i n e and o r i e n t e d specimens w i t h the l o n g m o l e c u l a r axes a l i g n e d n e a r l y p a r a l l e l t o each o t h e r . A t y p i c a l specimen d i f f r a c t s the i n c i d e n t x-ray beam t o g i v e a s e r i e s o f Bragg r e f l e c t i o n s sampled a t r e c i p r o c a l l a t t i c e p o i n t s on l a y e r l i n e s . The p o s i t i o n s o f the s p o t s depend on the u n i t c e l l d i m e n s i o n s . The d i s t r i b u t i o n o f i n t e n s i t i e s and the l a y e r l i n e s p a c i n g s r e v e a l the symmetry and p i t c h o f the h e l i x r e s p e c t i v e l y . Together w i t h the a c t u a l i n t e n s i t i e s o f the r e f l e c t i o n s , the s p a t i a l arrangement o f atoms i n the u n i t c e l l can be d e l i n e a t e d u s i n g computer model b u i l d i n g p r o c e d u r e s . Sampling o f x-ray d i f f r a c t i o n does not o c c u r w i t h u n i a x i a l l y o r i e n t e d (but n o n - c r y s t a l l i n e ) specimens which o n l y produce c o n t i n u o u s i n t e n s i t y d i s t r i b u t i o n on l a y e r l i n e s . I n f o r m a t i o n on i n t e r m o l e c u l a r a s s o c i a t i o n cannot be i n f e r r e d i n t h e s e c a s e s . Between the u n i a x i a l l y o r i e n t e d , n o n - c r y s t a l l i n e specimens and the o r i e n t e d p o l y c r y s t a l l i n e m a t e r i a l s , t h e r e i s a whole range o f " o r d e r i n g " which y i e l d s d i f f e r e n t types o f d i f f r a c t i o n p a t t e r n s i n terms o f d e t a i l e d s t r u c t u r e s ( 3 ) .


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MODEL BUILDING AND STRUCTURE REFINEMENT The f i r s t s t e p i n t h e s t r u c t u r e a n a l y s i s i s t o b u i l d a s t e r e o c h e m i c a l l y p l a u s i b l e model o f a m o l e c u l a r asymmetric u n i t t h a t f i t s into a helix. I t must have t h e p i t c h and symmetry c h a r a c t e r i s t i c s determined from the l a y e r l i n e s p a c i n g s and from t h e s y s t e m a t i c absences and o v e r a l l i n t e n s i t y d i s t r i b u t i o n i n t h e d i f f r a c t i o n p a t t e r n . The l i n k e d - a t o m t r e e geometry, based on t h e p r i m a r y s t r u c t u r e , i s s e t up t o d e s c r i b e t h e b u i l d i n g b l o c k o f t h e helix. A l l bond l e n g t h s and most bond a n g l e s a r e u s u a l l y f i x e d a t t h e i r expected s t a n d a r d v a l u e s which a r e d e r i v e d from a survey o f the c r y s t a l s t r u c t u r e s o f a p p r o p r i a t e monomers. The main v a r i a b l e s are the r e l e v a n t conformation angles. I f necessary, the e n d o c y c l i c bond a n g l e s i n t h e case o f f l e x i b l e pyranose r i n g s , and t h e g l y c o s i d i c bond a n g l e a t each b r i d g e oxygen atom, a r e a l s o v a r i e d . The Linked-Atom Least-Squares (LALS) procedure (4) developed i n o u r l a b o r a t o r y i s used f o r g e n e r a t i n g h e l i c a l s t r u c t u r e s . I t i s further used t o r e f i n e t h e h e l i c a l model a g a i n s t t h e x-ray i n t e n s i t i e s . The Bragg d a t a p e r m i t t h e r e f i n e m e n t o f the p a c k i n g parameters o f h e l i c e s i n the u n i t c e l l . The LALS program a l l o w s us t o a c h i e v e 1) g e n e r a t i o n o f c o m p r e s s i o n - f r e e h e l i c a l model which i s d e s i g n e d t o have the major c o n f o r m a t i o n a n g l e s i n p r e f e r r e d domains; 2) maximiza t i o n o f t h e agreement between observed and c a l c u l a t e d x-ray s t r u c t u r e a m p l i t u d e s ; and 3) o p t i m i z a t i o n o f nonbonded and hydrogen bond i n t e r a c t i o n s w i t h i n , and between m o l e c u l e s . I f t h e r e a r e competing models, t h e i r r e l a t i v e m e r i t s can be a s s e s s e d on t h e b a s i s o f any, o r a l l , o f the above t h r e e c r i t e r i a u s i n g H a m i l t o n ' s t e s t (5). In a d d i t i o n , t h e t r a d i t i o n a l c r y s t a l l o g r a p h i c R - f a c t o r can be used t o judge t h e c o r r e c t n e s s o f a model. In f a v o r a b l e c a s e s , where good q u a l i t y Bragg x-ray d a t a a r e a v a i l a b l e a s i n g e l l a n , p e r i o d i c water m o l e c u l e s and c a t i o n s i n t h e u n i t c e l l can be l o c a t e d from s u c c e s s i v e d i f f e r e n c e e l e c t r o n d e n s i t y maps. T h e i r p o s i t i o n s can be c o - r e f i n e d w i t h those o f t h e polymer c h a i n s u n t i l t h e guest m o l e c u l e s a r e p r o p e r l y c o o r d i n a t e d and/or hydrogen bonded w i t h t h e polymer c h a i n s and complete convergence i s reached. The LALS procedure has been s u c c e s s f u l l y employed t o s o l v e the t h r e e - d i m e n s i o n a l s t r u c t u r e s o f s e v e r a l polymers, u s i n g Bragg d i f f r a c t i o n d a t a from w e l l o r i e n t e d p o l y c r y s t a l l i n e f i b e r s . R e c e n t l y , a v e c t o r i z e d v e r s i o n (6) o f the LALS program f o r t h e supercomputer CYBER 205 has been implemented. T h i s has reduced t h e e x e c u t i o n time by more than an o r d e r o f magnitude and has t h e c a p a c i t y t o handle l a r g e asymmetric u n i t s c o n t a i n i n g up t o 1000 atoms o r more. The program i s coded t o i n c l u d e c o n t i n u o u s d i f f r a c t i o n a m p l i t u d e s as i n p u t d a t a . With the a v a i l a b i l i t y o f automated i n t e n s i t y measurement u s i n g t h e O p t r o n i c s f i l m scanner (Z>§), i t ^ p o s s i b l e t o use a m p l i t u d e s o f c o n t i n u o u s d i f f r a c t i o n and sampled, Bragg r e f l e c t i o n s e i t h e r s e p a r a t e l y o r j o i n t l y i n t h e r e f i n e m e n t o f h e l i c a l polymers. U s i n g these new p r o c e d u r e s , we have a l r e a d y determined t h e s t r u c t u r a l f e a t u r e s o f two DNA*RNA h y b r i d s ( 9 ) , t h r e e DNA-DNA d u p l e x e s (10) and t h e g e l f o r m i n g p o l y s a c c h a r i d e kappa-carrageenan ( J J ) , a l l o f which i n v o l v e c o n t i n u o u s i n t e n s i t i e s as d a t a s e t s . F u r t h e r d e t a i l s o f t h e procedure a r e g i v e n elsewhere ( 1 2 ) . s n

o

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MOLECULAR ARCHITECTURES I t i s worth p o i n t i n g o u t t h e p o t e n t i a l s o f computer m o d e l i n g v s . x-ray f i b e r d i f f r a c t i o n a n a l y s i s . Computer model b u i l d i n g e n a b l e s to g e n e r a t e a number o f p l a u s i b l e models, b u t i t cannot r e a d i l y d i s t i n g u i s h one from the o t h e r . X-ray f i b e r d i f f r a c t i o n a n a l y s i s i n v o l v e s computer m o d e l i n g , f o l l o w e d by l e a s t - s q u a r e s r e f i n e m e n t o f the s t r u c t u e a g a i n s t t h e e x p e r i m e n t a l l y measured x-ray d a t a . T h e r e f o r e , t h e merging o f modeling w i t h d i f f r a c t i o n d a t a h e l p s t o d i s c r i m i n a t e between v i a b l e a l t e r n a t i v e s and, i n f a v o r a b l e c a s e s , a l s o provides the best s o l u t i o n . The purpose o f t h i s paper i s t o i l l u s t r a t e t h i s w i t h a few r e c e n t examples. Here we summarize t h e important s t r u c t u r a l f e a t u r e s and t h e i r r e l a t i o n s h i p t o t h e o b s e r v e d p h y s i c a l p r o p e r t i e s o f g e l l a n and RTPS. The d e t e r m i n a t i o n o f t h e p o t a s s i u m g e l l a n c r y s t a l s t r u c t u r e was f a c i l i t a t e d by t h e excellent x-ray d a t a t o a f i n a l R-value o f 0.18. The a t o m i c c o o r d i n a t e s and s t r u c t u r e f a c t o r s have been l i s t e d by C h a n d r a s e k a r a n e t a l (J_3). T h i s s t r u c t u r e has been i n s t r u m e n t a l i n p r o d u c i n g a s p e c u l a t i v e c r y s t a l s t r u c t u r e o f c a l c i u m g e l l a n and t h e m o l e c u l a r s t r u c t u r e o f n a t i v e g e l l a n (J4_). T h i s i s an e x c e l l e n t example o f the accomplishment o f t h e model b u i l d i n g t e c h n i q u e . The p a u c i t y o f x-ray d a t a i n t h e case o f RTPS has n o t a l l o w e d us beyond t h e g e n e r a t i o n o f s i n g l e and d o u b l e - h e l i c a l models c o n s i s t e n t w i t h t h e h e l i c a l parameters d i s c e r n i b l e i n t h e d i f f r a c t i o n p a t t e r n . Since the d e t a i l s o f t h e c a l c u l a t i o n s have a l r e a d y been p u b l i s h e d , they a r e n o t r e p e a t e d i n t h i s paper. RHIZOBIUM TRIFOLII CAPSULAR POLYSACCHARIDE. This polysaccharide i s v e r y u n u s u a l i n t h a t i t has a doubly branched, n e u t r a l h e x a s a c c h a r i d e r e p e a t i n g u n i t (JT5) shown below. B-D-Gal-(4-1)6-D-Gal

1

6 -4)a-D-Glc(1-3)a-D-Man(1-3)6-D-Gal(12 t 1 a-D-Gal I t s aqueous s o l u t i o n s a r e n o t o n l y v i s c o u s b u t become g e l s o v e r t h e temperature range 20 - 90°C. The c o u r s e o f s o l - g e l t r a n s i t i o n a s a f u n c t i o n o f temperature m o n i t o r e d by o p t i c a l r o t a t i o n (_15) s u g g e s t s the c o n v e r s i o n o f a c o n f o r m a t i o n a l ^ m o b i l e random c o i l i n t h e s o l s t a t e t o a r i g i d o r d e r e d c o n f o r m a t i o n i n t h e g e l s t a t e . The m i d p o i n t o f t r a n s i t i o n i s a p p r o x i m a t e l y around 45°C. Only o r i e n t e d , n o n c r y s t a l l i n e f i b e r s c o u l d be p r e p a r e d , t h e b e s t o f them a t 4 5 ° C A t y p i c a l d i f f r a c t i o n p a t t e r n shown i n F i g u r e 1 i n d i c a t e s t h a t t h e m o l e c u l e s i n t h e specimen a r e a l i g n e d n e a r l y p a r a l l e l t o each o t h e r , but a r e n o t o r g a n i z e d l a t e r a l l y beyond a s h o r t range. The l a y e r l i n e s p a c i n g s a r e c o n s i s t e n t w i t h a c - r e p e a t o f 19.6Â. The f i r s t m e r i d i o n a l s p o t o c c u r s on t h e second l a y e r l i n e . The s t r o n g r e f l e c t i o n on t h e equator c o r r e s p o n d s t o 18.0Â s p a c i n g which m i r r o r s the l i k e l y d i a m e t e r o f the h e l i x .


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F i g u r e 1. An x-ray d i f f r a c t i o n p a t t e r n from an o r i e n t e d , n o n c r y s t a l l i n e f i b e r o f t h e Rhizobium trifolii capsular p o l y s a c c h a r i d e u s i n g CuKa r a d i a t i o n . (Reproduced w i t h p e r m i s s i o n from Ref. 16. C o p y r i g h t 1987 Gordon and Breach.)


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There a r e two d i s t i n c t s t e r e o c h e m i c a l p o s s i b i l i t i e s f o r t h e h e l i x which a r e c o n s i s t e n t w i t h t h e i n t e n s i t y d i s t r i b u t i o n . One o f them i s a 2 - f o l d s i n g l e - h e l i x o f p i t c h 19.6Â and the o t h e r a 4 - f o l d , h a l f - s t a g g e r e d , p a r a l l e l , d o u b l e - h e l i x o f p i t c h 39.2Â. The d o u b l e h e l i x c o u l d be r i g h t - o r l e f t - h a n d e d . In a l l c a s e s , t h e r e i s c o n s i d e r a b l e c o n f o r m a t i o n a l m o b i l i t y about t h e (1-*6) l i n k a g e o f t h e disaccharide side chain. P r e l i m i n a r y models have been b u i l t f o r each p o s s i b i l i t y and, due t o i n s u f f i c i e n t d i f f r a c t i o n d a t a , d e t a i l e d x-ray r e f i n e m e n t s have n o t been conducted f o r any o f them. The s i n g l e - h e l i x f o l l o w s a r i g h t - h a n d e d s i n u o u s path around t h e m o l e c u l a r a x i s a s shown i n F i g u r e 2. The d i s a c c h a r i d e s i d e c h a i n s t i c k s o u t ( F i g u r e 2a) o r f o l d s back on the main c h a i n ( F i g u r e 2b) c o r r e s p o n d i n g t o t h e c o n f o r m a t i o n θ[C4-C5-C6-06] b e i n g gauche plus or trans ( j_6). A r e p r e s e n t a t i v e s t r u c t u r e o f the d o u b l e - h e l i x (17) i s shown i n F i g u r e 3. In t h i s model, t h e s i d e c h a i n a d o p t s a f o l d e d conformation. Both s i n g l e - and d o u b l e - h e l i c e s i n d i c a t e a c o n s i d e r a b l e amount o f s i d e c h a i n — backbone i n t e r a c t i o n s and they may have a s t r u c t u r a l r o l e i n t h e g e l a t i o n p r o c e s s . These t e n t a t i v e s t r u c t u r e s cannot be d i s c r i m i n a t e d a t t h i s s t a g e . GELLAN. The e x t r a c e l l u l a r , a n i o n i c , l i n e a r p o l y s a c c h a r i d e from Pseudomonas elodea, i s c a l l e d g e l l a n i n i t s d e a c y l a t e d form. It i s i n d u s t r i a l l y important because o f i t s a b i l i t y t o form c a t i o n dependent g e l s . I t s t e t r a s a c c h a r i d e r e p e a t i n g m o t i f (A-B-C-D) i s g i v e n below: +3)B-D-Glc(1-4)6-D-GlcA(1-4)B-D-Glc(1-4)a-L-Rha(1*. S t r o n g and b r i t t l e g e l s can be formed w i t h g e l l a n i n t h e p r e s e n c e o f p o t a s s i u m o r a t a d r a s t i c a l l y reduced c o n c e n t r a t i o n o f c a l c i u m (J8i!9). N a t i v e g e l l a n , a s s e c r e t e d by the b a c t e r i u m , i s s u b s t i t u t e d by L - g l y c e r a t e groups a t C2 on a l l the 0-3 s u b s t i t u t e d g l u c o s e r e s i d u e s and a c e t a t e groups a t C6 on h a l f o f them (20) and i t forms o n l y weak and rubbery g e l s ( 2 ^ ) . Samples s u i t a b l e f o r d e t a i l e d s t r u c t u r e a n a l y s i s c o u l d be r e a d i l y p r e p a r e d from a number o f monovalent s a l t forms o f g e l l a n . A t y p i c a l d i f f r a c t i o n p a t t e r n from a w e l l o r i e n t e d , p o l y c r y s t a l l i n e f i b e r o f t h e p o t a s s i u m s a l t o f g e l l a n i s shown i n F i g u r e 4. S i m i l a r p a t t e r n s can be o b t a i n e d from the l i t h i u m form (22) and o t h e r monovalent s a l t s . The i n t e n s i t y d i s t r i b u t i o n s and u n i t c e l l dimensions (12) a r e v e r y s i m i l a r i n d i c a t i n g s t r u c t u r a l s i m i l a r i t y i n a l l these c a s e s . C r y s t a l s t r u c t u r e s o f the l i t h i u m (22) and p o t a s s i u m (13) forms have s o f a r been e s t a b l i s h e d . The l a t t e r has n o t o n l y c o n f i r m e d t h e m o l e c u l a r f e a t u r e s o f the g e l l a n d o u b l e - h e l i x f i r s t proposed ( 2 2 ) , but a l s o p r o v i d e d p r e c i s e d e t a i l s on t h e l o c a t i o n s o f c a t i o n s and water m o l e c u l e s and t h e i r i n t e r a c t i o n s w i t h the d o u b l e - h e l i c e s . F i g u r e 5 shows the morphology o f the d o u b l e - h e l i x which i s made up o f two h a l f - s t a g g e r e d , p a r a l l e l , 3 - f o l d l e f t - h a n d e d p o l y s a c c h a r i d e c h a i n s o f p i t c h 56.3Â. The major s t a b i l i z i n g f o r c e i s t h e i n t e r c h a i n hydrogen bond and t h e p o t a s s i u m c o o r d i n a t i o n , b o t h i n v o l v i n g t h e c a r b o x y l a t e group i n every t e t r a s a c c h a r i d e r e p e a t i n g unit. These i n t e r a c t i o n s , a s w e l l a s t h e i n t e r c h a i n hydrogen bonds, a r e i l l u s t r a t e d i n F i g u r e 6. Two d o u b l e - h e l i c e s a r e packed in an a n t i p a r a l l e l f a s h i o n i n t h e t r i g o n a l u n i t c e l l o f d i m e n s i o n s a = b = 15.8Â and c = 28.2Â w i t h a l a t e r a l s e p a r a t i o n o f 9.0Â


306


3.93 nm

Figure 2. Two mutually perpendicular of the 2-fold single-helix of RTPS. sidechain conformation is gauche plus and trans in (b). (Reproduced permission from Ref. 16. Copyright Gordon and Breach.)

views The in (a) with 1987

Figure 3. Two mutually perpendicular views of the 4-fold double-helix of RTPS.


18.



F i g u r e 4. X-ray d i f f r a c t i o n p a t t e r n from a w e l l o r i e n t e d and p o l y c r y s t a l l i n e f i b e r o f potassium g e l l a n u s i n g CuKa r a d i a t i o n . (Reproduced w i t h p e r m i s s i o n from Ref. 13. C o p y r i g h t 1988 Elsevier.)


308


F i g u r e 5. A s t e r e o view o f the g e l l a n d o u b l e - h e l i x f e a t u r i n g the i n t r a c h a i n hydrogen bonds ( t h i n dashed l i n e s ) , i n t e r c h a i n hydrogen bonds ( t h i c k dashed l i n e s ) , p o t a s s i u m i o n s ( f i l l e d c i r c l e s ) , and water m o l e c u l e s (open c i r c l e s ) and t h e s i x l i g a n d s a t t a c h e d t o each p o t a s s i u m i o n ( t h i n l i n e s ) .



F i g u r e 6. S c h e m a t i c i l l u s t r a t i o n o f the hydrogen-bonding and p o t a s s i u m i o n (K) c o o r d i n a t i o n i n t e r a c t i o n s i n the g e l l a n d o u b l e - h e l i x , i n c l u d i n g the f i r s t s h e l l water m o l e c u l e (W). The distances are given i n Â u n i t s .

310


(Figure 7). The g e l l a n m o l e c u l e s a r e c r o s s l i n k e d by d o u b l e - h e l i x — potassium — water — potassium — d o u b l e - h e l i x i n t e r a c t i o n s which a r e r e s p o n s i b l e f o r subsequent g e l a t i o n p r o c e s s . The p a c k i n g arrangement f u r t h e r i n d i c a t e s t h a t p a i r s o f c a r b o x y l a t e oxygen atoms b e l o n g i n g t o a d j a c e n t d o u b l e - h e l i c e s a r e 5.1Â a p a r t and t h a t the two p o t a s s i u m i o n s c o o r d i n a t e d t o t h e s e c a r b o x y l a t e s a r e 4.3Â from each o t h e r . As s p e c u l a t e d i n our p r e v i o u s study ( 1 3 ) , u s i n g computer model b u i l d i n g , we have been a b l e t o s u b s t i t u t e a s i n g l e c a l c i u m i o n , f o r the two p o t a s s i u m i o n s , which makes a d i r e c t c r o s s l i n k between the two d o u b l e - h e l i c e s (14) and t h i s i s shown i n F i g u r e 8. Such d i r e c t c r o s s l i n k s would be much s t r o n g e r than those o b s e r v e d i n the case o f p o t a s s i u m g e l l a n . This would e x p l a i n the s t r o n g g e l a t i o n b e h a v i o r o f c a l c i u m a t a v e r y s m a l l f r a c t i o n o f the c o n c e n t r a t i o n r e q u i r e d o f p o t a s s i u m o r o t h e r monovalent i o n s ( J J 3 , ] _ 9 ) . A view o f the proposed c r y s t a l s t r u c t u r e model f o r calcium g e l l a n i n the aib-plane i s shown i n Figure 9. NATIVE GELLAN. The computer model b u i l d i n g a p p r o a c h has f u r t h e r e n a b l e d us t o produce a s t e r e o c h e m i c a l l y s a t i s f a c t o r y m o l e c u l a r s t r u c t u r e o f n a t i v e g e l l a n ( j j l ) which i n c o r p o r a t e s the same g e l l a n d o u b l e - h e l i x backbone as found i n the p o t a s s i u m g e l l a n c r y s t a l s t r u c t u r e (_^3). The a c e t a t e group c o u l d be r e a d i l y a t t a c h e d t o t h e p e r i p h e r a l hydroxymethyl group o f the g l u c o s e r e s i d u e A i n e v e r y r e p e a t i n g m o t i f and t h i s a d d i t i o n does not p e r t u r b the p a c k i n g arrangement. On the o t h e r hand, the L - g l y c e r a t e group c o u l d be p r o p e r l y f i x e d o n l y a f t e r a m i l d r o t a t i o n o f 30° f o r the c a r b o x y l a t e group about i t s C5-C6 bond, a l o n g w i t h a c o n c e r t e d 2Â movement o f the p o t a s s i u m i o n and i t s f i r s t s h e l l water m o l e c u l e . Contrary to p r e v i o u s e x p e c t a t i o n s , the g l y c e r a t e group i s n o t o n l y accommodated i n the d o u b l e - h e l i x , but i t s secondary h y d r o x y l group a l s o c o n t r i b u t e s a d d i t i o n a l s t a b i l i t y by making an i n t e r c h a i n hydrogen bond w i t h the oxygen atom 02 o f the g l u c u r o n a t e r e s i d u e . The r e s u l t i n g morphology o f the n a t i v e g e l l a n d o u b l e - h e l i x w i t h p o t a s s i u m c o o r d i n a t i o n i s i l l u s t r a t e d i n F i g u r e 10. We found i t i m p o s s i b l e t o mimic the p o t a s s i u m g e l l a n p a c k i n g arrangement f o r the n a t i v e g e l l a n d o u b l e - h e l i c e s due t o s e v e r e i n t e r m o l e c u l a r s h o r t c o n t a c t s i n v o l v i n g the g l y c e r a t e groups and backbone atoms. These c o u l d not be r e l i e v e d w i t h o u t a s u b s t a n t i a l l a t e r a l e x p a n s i o n o f the u n i t c e l l which p a r t i c u l a r l y weakens t h e i n t e r m o l e c u l a r i n t e r a c t i o n s mediated by c a r b o x y l a t e g r o u p s , c a t i o n s and water m o l e c u l e s t h a t a r e c r u c i a l f o r m o l e c u l a r aggregation. T h i s might e x p l a i n the o b s e r v e d weak and rubbery g e l s o f t h i s material. CONCLUSIONS Computer model b u i l d i n g and x-ray f i b e r d i f f r a c t i o n s t u d i e s on the Rhizobium t r i f o l i i c a p s u l a r p o l y s a c c h a r i d e and g e l l a n have p r o v i d e d the m o l e c u l a r g e o m e t r i e s o f some newly d e v e l o p e d g e l - f o r m i n g p o l y s a c c h a r i d e s u s e f u l t o the food i n d u s t r y . Preliminary singleand d o u b l e - h e l i c a l models o f RTPS would be s t a b i l i z e d p r i m a r i l y by side chain — backbone i n t e r a c t i o n s . The c r y s t a l s t r u c t u r e o f p o t a s s i u m g e l l a n shows t h a t the m o l e c u l e i s a h a l f - s t a g g e r e d , p a r a l l e l d o u b l e - h e l i x s t a b i l i z e d by i n t e r c h a i n hydrogen bonds and


18. CHANDRASEKARAN & THAILAMBAL


2B15nm

Figure 7. Packing of two adjacent, up and down, gellan molecules, the potassium ions (filled circles) and the water molecules (open circles) coordinated to them in the unit cell, viewed along the [110] direction. (Reproduced with permission from Ref. 13. Copyright 1988 Elsevier.)

2.815 nm

Figure 8. Proposed packing arrangement of calcium gellan, showing two, up and down, double-helices crosslinked at arrows by calcium ions (filled circles) and associated water molecules (open circles) in the unit cell, viewed normal to the molecular axes. (Reproduced with permission from Ref. 14. Copyright 1989 Elsevier.)


312


Figure 9. The c r y s t a l s t r u c t u r e model f o r c a l c i u m g e l l a n viewed a l o n g t h e c - a x i s . The c a l c i u m i o n s a r e shown by f i l l e d c i r c l e s and water m o l e c u l e s by open c i r c l e s . (Reproduced w i t h p e r m i s s i o n from Ref. 14. C o p y r i g h t 1989 E l s e v i e r . )


18. CHANDRASEKARAN & THAILAMBAL


Figure 10. A stereo view of the native gellan double-helix and the coordinating potassium ions ( f i l l e d c i r c l e s ) and water molecules (open c i r c l e s ) . The intrachain (thin dashed l i n e s ) , interchain (thick dashed l i n e s ) hydrogen bonds and ligands to potassium ions (thin l i n e s ) s t a b i l i z e the double-helix. Both acetate and glycerate groups are shown along with their hydrogen atoms. (Reproduced with permission from Ref. 14. Copyright 1989 E l s e v i e r . ) via coordination of potassium ions with the carboxylate groups. A n t i p a r a l l e l packing arrangement of double-helices leads to the aggregation of molecules and subsequent gelation process. Extrapolation of the potassium c r y s t a l structure using model building techniques has enabled us to v i s u a l i z e the most probable calcium gellan c r y s t a l structure which explains i t s strong g e l l i n g behavior a t very low concentrations. Native gellan which has L-glycerate and acetate substitutions can also exist as a doubleh e l i x . Its weak g e l l i n g behavior apparently i s due to i t s i n a b i l i t y to retain the tight packing arrangement observed for the deacetylated material. ACKNOWLEDGMENTS We thank Kelco (1271670), National Science Foundation (8606942) and the Industrial Consortium of the Whistler Center for f i n a n c i a l support and Becky H i t t for word processing. French and Brady; Computer Modeling of Carbohydrate Molecules ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

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Literature Cited 1. Arnott, S. In Developments in Food Carbohydrate; Birch, G.G.; Shallenberger, R.S., Eds.; Applied Science Publishers: England, 1977; pp 43-60. 2. Arnott, S.; Chandrasekaran, R. In Biopolymeri; Proceedings of the Italian Association for the Sicence and Technology of Macromolecules Summer School, Cargnano di Garcia, Italy, 1984; XXIII 1-12. 3. Arnott, S. In Fiber Diffraction Methods; French, A.D.; Gardner, K.H., Eds.; ACS Symposium Series No. 14; American Chemical Society: Washington, D.C., 1980; pp 1-30. 4. Smith, P.J.C; Arnott, S. Acta Crystallogr. 1978, A34, 3-11. 5. Hamilton, W.C. Acta Crystallogr. 1965, 18, 502-10. 6. Millane, R.P.; Byler, M.A.; Arnott, S. In Supercomputer Applications; Numrich; R.W., Ed.; Plenum Publishers: New York, 1985; pp 289-302. 7. Millane, R.P.; Arnott, S. J . Appl. Crystallogr. 1985, 18, 419-23. 8. Millane, R.P.; Arnott, S. J . Macromol. S c i . , Physics 1985, B24, 193-227. 9. Arnott, S.; Chandrasekaran, R.; Millane, R.P.; Park, H.-S. Mol. Biol. 1986, 188, 631-40. 10. Park, H.-S.; Arnott, S.; Chandrasekaran, R.; Millane, R.P.; Campagnari, F. J . Mol. Biol. 1987, 197, 513-23. 11. Millane, R.P.; Chandrasekaran, R.; Arnott, S.; Dea, I.C.M. Carbohydr. Res. 1988, 182, 1-17. 12. Chandrasekaran, R. In Frontiers in Carbohydrate Research: Food Applications; Millane, R. P.; BeMiller, J . N . ; Chandrasekaran, R., Eds.; Elsevier: New York, 1989, in press. 13. Chandrasekaran, R.; Puigjaner, L . C . ; Joyce, K . L . ; Arnott, S. Carbohydr. Res. 1988, 181, 23-40. 14. Chandrasekaran, R.; Thailambal, V.G. Carbohydr. Polymers 1989, in press. 15. Gidley, M.J.; Dea, I.C.M.; Eggleston, G.; Morris, E.R. Carbohydr. Res. 1987, 160, 381-96. 16. Chandrasekaran, R.; Millane, R.P.; Walker, J . K . ; Arnott, S.; Dea, I.C.M. In Industrial Polysaccharides: The Impact of Biotechnology and Advanced Methodologies; Stivala, S.S.; Crescenzi, V.; Dea, I.C.M., Eds.; Gordon and Breach: New York, 1987; pp 111-8. 17. Chandrasekaran, R.; Millane, R.P.; Arnott, S. In Gums and Stabilisers for the Food Industry 4; Phillips, G.O.; Wedlock, D . J . ; Williams, P.Α., Eds.; IRL Press: Oxford, 1988; pp 18391. 18. Crescenzi, V.; Dentini, M.; Coviello, T. Carbohydr. Res. 1986, 149, 425-32. 19. Grasdalen, H.; Smidsrod, O. Carbohydr. Polymers 1987, 7, 371-94. 20. Kuo, M.-S.; Dell, Α.; Mort, A.J. Carbohydr. Res. 1986, 156,173-87. 21. Moorhouse, R. In Industrial Polysaccharides: Genetic Engineering, Structure/Property Relations and Applications; Yalpani, M., Ed.; Elsevier: Amsterdam, 1987; pp 187-206. 22. Chandrasekaran, R.; Millane, R.P.; Arnott, S.; Atkins, E.D.T. Carbohydr. Res. 1988, 175, 1-15. RECEIVED February 13, 1990


A New Generation of Gel-Forming Polysaccharides - ACS Symposium

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